Without limiting the scope of the invention, its background is described in connection with stochastic sensing based on the detection of individual binding events between analyte molecules and a single sensor element. Upon binding, a property of the sensor element is altered. This property or the effects of the changed property are measured.
In a simple example, the sensor element is a protein that is altered when it binds another molecule. The binding molecule to be detected is referred to as the analyte. The alteration of the sensor element that occurs upon binding is measured either directly or indirectly. In simple systems the alteration produces a simple signal, such as a difference in electrical current, force or fluorescence. Measurements of the signal indicate whether the analyte is bound and how long it remains bound. The frequency of occurrence of binding events is determined by the concentration of the analyte. The nature of the binding event is determined by the binding properties of the analyte, which determine, for example, the magnitude and duration of the resulting signal. Thus, a single sensor element to which multiple analytes may bind either directly may be used to determine which of those analytes are in a solution and the concentration of each particular analyte.
Protein pores are being developed for use in stochastic sensing.[1] For several applications, the pores must be stable at high temperatures. The structures of many integral membrane proteins remain intact at high temperatures[2] and the existence of extremophiles implies that membrane proteins function at 100° C. and beyond.[3] However, few measurements of membrane protein activity have been made at temperatures above ˜55° C., and none have been made for ion channels and pores.
The photocycle of bacteriorhodopsin, for example, has been examined at temperatures up to 85° C.[4] The activity is compromised above 60° C. because the chromophore, a transretinal Schiff's base, isomerizes to the 13-cis form. Transmembrane proton pumping was not measured directly in these experiments. Macroscopic current recordings with valinomycin (a macro cyclic antibiotic) have been carried out at up to 80° C. in bilayers comprising lipids from a hyperthermophile, Caldariella acidophila.[5]
In the case of proteinaceous channels and pores, activity has been measured at up to ˜55° C. For example, multichannel recordings of vanilloid receptors (temperature-sensitive cation channels) have been made after expression in Xenopus oocytes, and several subtypes of the receptor remain active at 55° C.[6] Recently, single channel recordings of the vanilloid receptor VR1 have been made at up to 55° C.[7] The transmembrane domains of these proteins are presumably largely α-helical.
Stochastic sensing may be accomplished with various sensing elements, using various modes of detection. One simple model uses an ion channel protein pore embedded in a membrane between a cis chamber and a trans chamber. When the pore is fully open a large ion flux occurs (e.g., 108 ions/s) which constitutes an electrical current that may be monitored by single channel recording. When an analyte binds to the pore, ion flux is altered, usually by decreasing the flow of ions. This generates a current trace that shows conduction over time.
One particular pore that has been used in stochastic sensing is Staphlococcus alpha hemolysin (αHL), which is actually an exotoxin secreted by Staphylococcus aureus. The monomeric 293 amino acid polypeptide can self-assemble on lipid bilayers, such as membranes, to form a heptameric pore. Alternatively, pre-formed pores may be inserted into a lipid bilayer. The pore is a mushroom-shaped structure in which the lower half of the stem forms a transmembrane channel. The interior of the pore is referred to as the “lumen” and may be accessible from outside the pore. By convention, when the pore is situated in a membrane, the side of the membrane on which the top of the mushroom shape is located is designated as the “cis” side of the membrane. The side of the membrane to which the stem portion leads is designated the “trans” side of the membrane. The pore essentially forms a hole in the membrane through which ions will flow if an electric potential is generated between the two chambers.
Measurements on the pore-forming toxin, α-hemolysin, one of the proteins examined in the present application, have been made previously at up to 50° C.[8-10] This protein largely includes β structures. Indeed, all three of the proteins examined here contain β barrels, which are formed from either a single subunit, OmpG; seven subunits, α-hemolysin (αHL); or eight subunits, leukocidin (Luk). OmpG is a 280-residue polypeptide that forms a β barrel of, most likely, 16 antiparallel strands. Unlike most porins, which are trimeric, OmpG functions as a monomer.[11] The homoheptameric pore formed by αHL is a mushroom-shaped structure.[12] The stem of the mushroom is a 14-stranded transmembrane β barrel with two strands contributed by each subunit. The stem is capped by a large hollow extracellular domain. The Luk pore contains two types of subunits, F and S, which are related in sequence and structure to αHL.[13,14] The pore is a heterooctamer containing four F and four S subunits.[15] Despite the presence of only one additional subunit, the unitary conductance of the Luk pore is more than three times that of the αHL pore.[16]